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Originally published In Press as doi:10.1074/jbc.M209493200 on October 16, 2002

J. Biol. Chem., Vol. 277, Issue 52, 50693-50702, December 27, 2002
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Covalent Modification of Epithelial Fatty Acid-binding Protein by 4-Hydroxynonenal in Vitro and in Vivo
EVIDENCE FOR A ROLE IN ANTIOXIDANT BIOLOGY*

Assumpta Bennaars-EidenDagger , LeeAnn HigginsDagger , Ann V. HertzelDagger , Rebecca J. Kapphahn§, Deborah A. Ferrington§, and David A. BernlohrDagger

From the Departments of Dagger  Biochemistry, Molecular Biology and Biophysics, and § Ophthalmology, University of Minnesota, Minneapolis, Minnesota 55455

4-Hydroxynonenal (4-HNE) is a cytotoxic alpha ,beta -unsaturated acyl aldehyde that is naturally produced from lipid peroxidation and cleavage in response to oxidative stress and aging. Such reactive lipids covalently modify cellular target proteins, thereby affecting biological structure and function. Herein we report the identification of the epithelial fatty acid-binding protein (E-FABP) as a molecular target for 4-HNE modification both in vitro and in vivo. 4-HNE covalently modified (t1/2 < 60 s) E-FABP in vitro, as revealed by a combination of matrix-assisted laser desorption ionization-time of flight mass spectrometry and immunochemical reactivity using antibodies directed to 4-HNE-protein conjugates. Identification of Cys-120 as the major site of modification was determined through tandem mass spectral sequencing of tryptic peptides, as well as analysis of E-FABP mutants C120A, C127A, and C120A/C127A. The in vitro modification of Cys-120 by 4-HNE was relatively insensitive to pH (6.4-8.4), and temperature (4-37 °C) but was markedly potentiated by noncovalently bound fatty acids. 4-HNE-modified E-FABP was more stable than unmodified E-FABP to chemical denaturation by guanidine hydrochloride, as assessed by changes in intrinsic tryptophan fluorescence. Analysis of soluble protein extracts from rat retina with antibodies directed to 4-HNE-protein conjugates revealed immunoreactivity with a 15-kDa protein that was identified by electrospray ionization and matrix-assisted laser desorption ionization-time of flight mass spectrometry as E-FABP. Evaluation of retinal pigment epithelial cell extracts derived from E-FABP null mice by two-dimensional gel electrophoresis using anti-4-HNE antibodies revealed increased modification in the null cells relative to those from wild type cells. These results indicate that E-FABP is a molecular target for 4-HNE modification and the hypothesis that E-FABP functions as an antioxidant protein by scavenging reactive lipids through covalent modification of Cys-120.

* This work was supported by National Institutes of Health NIA Grant RO3 AG19024, by the Foundation Fighting Blindness, by the American Federation of Aging Research, by an unrestricted grant to the Department of Ophthalmology from the Research to Prevent Blindness Foundation (to D. A. F.), and by a grant from the National Science Foundation (to D. A. B.). A. B.-E. was supported by NIH NHLBI Grant T32HL07741.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, 321 Church St. S.E., Minneapolis, MN 55455. Tel.: 612-624-2712; E-mail: bernl001@umn.edu.

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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